A primer design method and primer set

Abstract

This invention discloses a primer design method and primer set. Through in-depth analysis of influenza surveillance network data nationwide, this application makes significant contributions to improving the accuracy and sensitivity of influenza virus identification. The primer revisions not only achieved significant results in experiments but also provided valuable insights for future influenza surveillance and research. A deeper understanding of the genetic characteristics and epidemiological patterns of influenza viruses is crucial for developing scientifically effective prevention and control strategies. In disease prevention and public health management, continuous improvement of surveillance methods and strategies will bring greater benefits to timely responses to infectious diseases such as influenza.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a primer design method and primer set. Background Technology

[0002] Currently, when using polymerase chain reaction (PCR) technology for gene capture, the primers used are designed based on the deoxyribonucleic acid (DNA) sequence of a reference genome. This reference genome DNA sequence was detected through the Human Genome Project and includes the genomic genes of most organisms. However, due to varying degrees of mutation in the reference genome, primer sensitivity is reduced. Therefore, designing universal primers is crucial in this field. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention provides a primer design method and primer set.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A first aspect of the present invention provides a primer design method, the method comprising:

[0006] Obtain a target gene database, wherein the target gene database includes target gene sequences;

[0007] Screening for target gene mutation sites;

[0008] Primers are designed based on the target gene mutation site, which includes one or more gene mutation sites and / or degenerate bases.

[0009] Furthermore, the primers are influenza virus primers.

[0010] Furthermore, the influenza virus primers are H3 influenza virus primers.

[0011] Furthermore, the target gene mutation site includes one or more of c.308T>C, c.357T>A, c.317A>G, c.364A>G, and c.389A>G.

[0012] Furthermore, the degenerate bases include one or more of R1 at site 317, R2 at site 364, or Y.

[0013] In this context, the degenerate base R represents A or G, and the degenerate base Y represents C or T.

[0014] A second aspect of the present invention provides a primer set for detecting influenza virus, the primer set comprising sequences having at least 90% identity with any one of the primer pairs shown in SEQ ID NO:4 and 6, SEQ ID NO:7 and 9, SEQ ID NO:10 and 12, SEQ ID NO:13 and 15, SEQ ID NO:16 and 18, SEQ ID NO:19 and 21, SEQ ID NO:22 and 24, SEQ ID NO:25 and 27, SEQ ID NO:28 and 30, SEQ ID NO:31 and 33, SEQ ID NO:34 and 36, SEQ ID NO:37 and 39, SEQ ID NO:40 and 42, SEQ ID NO:43 and 45, and SEQ ID NO:46 and 48.

[0015] Furthermore, the primer set includes any one of the primer pairs shown in SEQ ID NO:4 and 6, SEQ ID NO:7 and 9, SEQ ID NO:10 and 12, SEQ ID NO:13 and 15, SEQ ID NO:16 and 18, SEQ ID NO:19 and 21, SEQ ID NO:22 and 24, SEQ ID NO:25 and 27, SEQ ID NO:28 and 30, SEQ ID NO:31 and 33, SEQ ID NO:34 and 36, SEQ ID NO:37 and 39, SEQ ID NO:40 and 42, SEQ ID NO:43 and 45, and SEQ ID NO:46 and 48.

[0016] Furthermore, the primer set is selected from the primer pairs shown in SEQ ID NO:16 and 18 and / or the primer pairs shown in SEQ ID NO:37 and 39.

[0017] Furthermore, the primer set is selected from the primer pairs shown in SEQ ID NO:37 and 39.

[0018] Furthermore, the primer set also includes probes.

[0019] Furthermore, the probe comprises a sequence having at least 90% identity with any one of the sequences shown in SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, and SEQ ID NO:47.

[0020] Furthermore, the probe includes any one of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, and SEQ ID NO:47.

[0021] Furthermore, the probe is selected from EQ ID NO:17 and / or SEQ ID NO:38.

[0022] Furthermore, the probe is selected from SEQ ID NO:38.

[0023] Furthermore, the influenza virus primers are H3 influenza virus primers.

[0024] Furthermore, the primer pair includes a forward primer and a reverse primer.

[0025] Furthermore, at least one of the forward and reverse primers is labeled with a labeling substance.

[0026] Furthermore, the labeling substance includes fluorescent substances, radioactive isotopes, or enzymes.

[0027] Furthermore, the fluorescent material includes TAMRAT. TM Alexa555, Alexa647, Cy3 and Cy5 from the cyanine dye series, and fluorescein.

[0028] Furthermore, the radioactive isotope includes 32 P, 33 P, 35 S.

[0029] Furthermore, the enzymes include alkaline phosphatase and horseradish peroxidase.

[0030] A third aspect of the present invention provides a kit for detecting influenza virus in a sample, the kit comprising the primer set described in the second aspect of the present invention.

[0031] Furthermore, the kit also includes enzymes for PCR, dNTPs, buffer solutions, and Mg2+. 2+ At least one reactant in the mixture.

[0032] Furthermore, the enzymes used for PCR include DNA polymerase and / or RNA polymerase.

[0033] Furthermore, the buffer solution includes polymerase buffer and Tris hydrochloric acid buffer.

[0034] Furthermore, the kit also includes instructions.

[0035] Furthermore, the influenza virus primers are H3 influenza virus primers.

[0036] A fourth aspect of the present invention provides a method for detecting influenza virus in a sample, the method comprising amplification using the primer set described in the second aspect of the present invention.

[0037] Furthermore, the influenza virus primers are H3 influenza virus primers.

[0038] Furthermore, the method described is not for diagnostic purposes.

[0039] The fifth aspect of the present invention provides the use of the primer set described in the second aspect of the present invention in the preparation of a kit for detecting influenza virus.

[0040] Furthermore, the influenza virus primers are H3 influenza virus primers.

[0041] A sixth aspect of the present invention provides a primer design system, the system comprising:

[0042] Acquisition unit: used to acquire a target gene database, the target gene database including target gene sequences;

[0043] Mutation site screening unit: used to screen for mutation sites in target genes;

[0044] Primer design unit: used to design primers based on target gene mutation sites, which include one or more gene mutation sites and / or degenerate bases.

[0045] Furthermore, the primers are influenza virus primers.

[0046] Furthermore, the influenza virus primers are H3 influenza virus primers.

[0047] Furthermore, the target gene mutation site includes one or more of c.308T>C, c.357T>A, c.317A>G, c.364A>G, and c.389A>G.

[0048] Furthermore, the degenerate bases include one or more of R1 at site 317, R2 at site 364, or Y.

[0049] In this context, the degenerate base R represents A or G, and the degenerate base Y represents C or T.

[0050] A seventh aspect of the present invention provides an electronic device including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor, when executing the computer program, performs the steps of the method as described in the first aspect of the present invention.

[0051] Advantages and beneficial effects of the present invention:

[0052] This application, through in-depth analysis of data from a nationwide influenza surveillance network, makes significant contributions to improving the accuracy and sensitivity of influenza virus identification. The primer revisions not only yielded significant results in experiments but also provided valuable insights for future influenza surveillance and research. A deeper understanding of the genetic characteristics and epidemiological patterns of influenza viruses is crucial for developing scientifically effective prevention and control strategies. In disease prevention and public health management, continuous improvement of surveillance methods and strategies will bring greater benefits to timely responses to infectious diseases such as influenza. Attached Figure Description

[0053] Figure 1 This is a flowchart of primer design;

[0054] Figure 2 This is a map of variant sites 317, 364, and 389;

[0055] Figure 3 It is a 16-pair primer design roadmap;

[0056] Figure 4 It is a statistical chart of the maximum and minimum values ​​contained in 16 primer pairs;

[0057] Figure 5 This is a statistical graph of the arithmetic mean and variance of the alignment results of 15 primer pairs;

[0058] Figure 6 It is a distribution diagram of the aggregation and dispersion of primers. Detailed Implementation

[0059] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0060] In some processes described in the specification, claims, and accompanying drawings of this application, multiple operations appearing in a specific order are included. However, it should be clearly understood that these operations may not be performed in the order they appear herein, or may be performed in parallel. The operation numbers, such as 101, 102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be performed sequentially or in parallel. It should be noted that the descriptions such as "first," "second," etc., in this application are used to distinguish different messages, devices, modules, etc., and do not represent a sequential order, nor do they limit "first" and "second" to different types.

[0061] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0062] Figure 1 This is a flowchart of primer design, and the specific process is shown below:

[0063] 101: Obtain the target gene database;

[0064] 102: Screening for target gene mutation sites;

[0065] 103: Design primers based on the target gene mutation site.

[0066] In one embodiment, the target gene database is a collection of target genes, which can be any sequence. In a preferred embodiment, the target gene is a strain sequence, which can include the evolutionary process of the strain within a certain period and range, i.e., any strain sequence that has undergone mutation. In a more preferred embodiment, the strain sequence is an influenza virus sequence. In a specific embodiment, the influenza virus sequence is an H3 subtype influenza virus sequence.

[0067] In one embodiment, the target gene mutation site can be any mutation site of the selected target gene. In a specific embodiment, the H3 subtype influenza virus mutation site includes one or more of c.308T>C, c.357T>A, c.317A>G, c.364A>G, and c.389A>G.

[0068] For example, primers can be designed by mutating the sequence c.308T>C and c.357T>A, denoted as primer 1; primers can be designed by mutating c.317A>G based on c.308T>C and c.357T>A, denoted as primer 2; primers can be designed by mutating c.364A>G based on c.308T>C and c.357T>A, denoted as primer 3; primers can be designed by mutating c.389A>G based on c.308T>C and c.357T>A, denoted as primer 4; primers can be designed by mutating c.317A>G and c.364A>G based on c.308T>C and c.357T>A, denoted as primer 5. 5) Based on c.308T>C and c.357T>A, mutations of c.364A>G and c.389A>G can be performed, and primers can be designed, denoted as primer 6; based on c.308T>C and c.357T>A, mutations of c.317A>G and c.389A>G can be performed, and primers can be designed, denoted as primer 7; based on c.308T>C and c.357T>A, mutations of c.317A>G, c.364A>G, and c.389A>G can be performed, and primers can be designed, denoted as primer 8; based on c.308T>C and c.357T>A, a degenerate base R1 (A / G) can be added at position 317, and primers can be designed, denoted as primer 9. 9) Primers can be designed by adding a degenerate base R2 (A / G) at position 364 based on c.308T>C and c.357T>A, and designated as primer 10; primers can be designed by adding a degenerate base Y (C / T) at position 308 based on c.308T>C and c.357T>A, and designated as primer 11; primers can be designed by adding a degenerate base R1 (A / G) at position 317 and a degenerate base R2 (A / G) at position 364 based on c.308T>C and c.357T>A, and designated as primer 12; primers can be designed by adding a degenerate base R1 (A / G) at position 317 and a degenerate base Y (C / T) at position 308 based on c.308T>C and c.357T>A, and designated as primer 13. 13); Based on c.308T>C and c.357T>A, degenerate base R2 (A / G) can be added at position 364 and degenerate base Y (C / T) can be added at position 308 to design primers, denoted as primer 14; primers can be designed based on c.308T>C and c.357T>A.Based on the 357T>A sequence, degenerate base R1 (A / G) was added at position 317, degenerate base R2 (A / G) at position 364, and degenerate base Y (C / T) at position 308. Primers were designed and designated as primer 15.

[0069] The present invention provides a primer set for detecting influenza virus, wherein the primer set has at least 90% sequence identity with any of the primer pairs shown above.

[0070] In one implementation, a primer refers to a short nucleic acid molecule. A hybrid can be formed between the primer and the target nucleic acid strand by annealing with a complementary target nucleic acid molecule through nucleic acid hybridization. The primer can be extended along the target nucleic acid molecule using polymerase. Therefore, primers can be used to amplify target nucleic acid molecules, wherein the primer sequence is specific to the target nucleic acid molecule, so that, for example, the primer will hybridize with the target nucleic acid molecule under very high hybridization tightness conditions.

[0071] In one implementation, primers and primer pairs are used interchangeably, with each primer pair comprising at least one forward primer and at least one reverse primer, wherein the primers are specific for the amplification of influenza virus nucleic acid molecules. The forward or upstream primer is the primer oriented relative to the 5' direction of a reference site on the nucleic acid sequence; the reverse or downstream primer is the primer oriented relative to the 3' direction of a reference site on the nucleic acid sequence. Generally, at least one forward and one reverse primer are included in the amplification reaction.

[0072] At least one of the forward and reverse primers is labeled with a labeling substance.

[0073] The labeling substances include, but are not limited to, fluorescent substances, radioactive isotopes, or enzymes.

[0074] Fluorescent substances include, but are not limited to, TAMRAT TM Alexa555, Alexa647, Cy3 and Cy5 from the cyanine dye series, and fluorescein.

[0075] Radioactive isotopes include, but are not limited to, 32 P, 33 P, 35 S.

[0076] Enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.

[0077] In the primers of this application labeled with a labeling substance, the labeling substance can bind directly to the primer or bind via a linker. As the linker, any linker commonly used in this field is acceptable; specifically, for example, a nucleic acid of 1 to 3 bases is preferred, DNA of 1 to 3 bases is more preferred, DNA of 2 bases is even more preferred, and adenine (A)-adenine (A) is particularly preferred.

[0078] As for the method of labeling the primers involved in this application with fluorescent substances, it is sufficient to follow the methods known in the art, specifically, for example, the method of incorporating nucleotides labeled with fluorescent dye into primers according to the methods known in the art.

[0079] As for the method of labeling the primers involved in this application with radioactive isotopes, any method known in the art can be used. Specifically, examples include methods of labeling by incorporating a nucleotide labeled with a radioactive isotope. Specifically, examples include random primer methods, nick-shifting methods, 5' end labeling based on T4 polynucleotide kinase, and 3' end labeling based on terminal deoxynucleotidyl transferase. The primer set includes at least one modified nucleotide.

[0080] As for the method of labeling the primers involved in this application with enzymes, it is sufficient to follow any well-known method commonly practiced in the field. Specifically, examples include direct labeling methods, such as directly covalently binding enzyme molecules like alkaline phosphatase or horseradish peroxidase to the primers to be labeled.

[0081] In one embodiment, identity / similarity refers to the similarity or identity between two or more nucleic acid sequences, or two or more amino acid sequences, expressed based on the identity or similarity between sequences. Sequence identity can be measured by percentage identity; the higher the percentage, the more consistent the sequences. When aligned using standard methods, homologs or orthologs of nucleic acid or amino acid sequences have a relatively high level of sequence identity / similarity. The primers disclosed in this application are not limited to the exact sequences shown, and as those skilled in the art will recognize, the sequences can be altered if necessary without significantly affecting the primers' ability to function.

[0082] The present invention provides a kit for detecting influenza virus in a sample, the kit comprising the above-mentioned primer set.

[0083] In one embodiment, suitable amounts of one or more primers are provided in one or more containers or immobilized on a matrix. The primers may be provided as a suspension in an aqueous solution or, for example, as a lyophilized or freeze-dried powder. The container providing the nucleic acid can be any conventional container capable of containing the provided form, such as a microcentrifuge tube, ampoule, or bottle. The kit may contain labeled or unlabeled probes for detecting influenza virus nucleotide sequences.

[0084] In some applications, one or more primers (as described above) can be provided in pre-measured, single-use amounts in separate, typically disposable tubes or equivalent containers. Using such a setup, samples for testing the presence of influenza virus can be added to separate tubes for direct amplification.

[0085] The amount of nucleic acid primers provided in the kit can be any suitable amount, depending on the target market for the product. For example, if the kit is intended for research or clinical applications, the amount of each nucleic acid primer provided can be sufficient to initiate several PCR amplification reactions. General guidelines for determining suitable amounts can be found in the literature of Innis et al., Sambrook et al., and Ausubel et al. The kit may contain more than two primers to facilitate PCR amplification of larger quantities of influenza virus nucleotide sequences.

[0086] In some embodiments, the kit may contain reaction reagents necessary for performing PCR amplification, including DNA sample preparation reagents, enzymes for PCR, buffers, and Mg2+. 2+ and deoxyribonucleotides (dNTPs).

[0087] The enzymes used in PCR include DNA polymerase and / or RNA polymerase.

[0088] The DNA polymerases include, but are not limited to, Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, and the Klenow fragment.

[0089] The buffer solution is used to adjust the pH of the sample provided for PCR analysis to 7.0–10.0 (more preferably pH 8.0–9.0).

[0090] Buffer solutions include, but are not limited to, polymerase buffer and Tris hydrochloride buffer.

[0091] dNTPs are nucleoside sources for PCR-based DNA amplification; dATP, dGTP, dCTP, and dTTP are essential. Additionally, for dNTPs, chemically modified substances designed for hot-start methods can be used, such as CleanAmp from TriLink BioTechnologies, Inc. TM dNTP.

[0092] In PCR-based DNA amplification, Mg 2+ It is necessary. As Mg 2+ Sources include, but are not limited to, MgCl2, MgSO4, etc. MgCl2 is preferred.

[0093] The kit also includes fluorescent dyes.

[0094] In one embodiment, various known fluorescent dyes can be used. Examples include methods using intercalators with marking functions, and methods using probes that bind fluorescent substances to nucleotides that specifically hybridize to relatively amplified DNA sequences. Examples of intercalators include ethidium bromide and SYBR Green I as unsaturated fluorescent dyes, and Resolight and EvaGreen as saturated fluorescent dyes. Preferred intercalators are SYBR Green I as an unsaturated fluorescent dye, and EvaGreen and Resolight as saturated fluorescent dyes; more preferably, EvaGreen and Resolight are saturated fluorescent dyes. The amount used is determined according to the recommendations of the manufacturer or distributor of the fluorescent dye used.

[0095] The kit also includes an instruction manual, which may include guidance on obtaining and processing samples.

[0096] In addition, the kit may contain bacterial genomic DNA as a positive control for PCR and sterile water as a negative control.

[0097] When implementing this application, as other necessary equipment, examples include pipettes, pipette tips, 1.5ml microtubes, etc., which are widely used in molecular biology experiments. As for devices, examples include PCR machines, clean benches, tube centrifuges, etc., which are widely used in molecular biology experiments.

[0098] This invention provides a primer design system, the system comprising:

[0099] Acquisition unit: used to acquire a target gene database, the target gene database including target gene sequences;

[0100] Mutation site screening unit: used to screen for mutation sites in target genes;

[0101] Primer design unit: used to design primers based on target gene mutation sites, which include one or more gene mutation sites and / or degenerate bases.

[0102] In one embodiment, the system includes a processor, which may be a single-core or multi-core processor, or more than one processor for parallel processing. The system also includes memory (e.g., random access memory, read-only memory, flash memory), electronic storage units (e.g., hard disks), communication interfaces (e.g., network adapters) for communicating with one or more other systems, and peripheral devices such as cache memory, other memory, data storage, and / or electronic display adapters. The memory, electronic storage units, communication interfaces, and peripheral devices communicate with the processor via a communication bus (solid line), such as a motherboard. The storage units may be data storage units (or databases) for storing data. The system may be operatively coupled to a computer network by means of the communication interface. The network may be the Internet, an intranet and / or an extranet, or an intranet and / or extranet communicating with the Internet. In some cases, the network is a communication and / or data network. The network may include one or more computer servers, which may support distributed computing, such as cloud computing. In some cases, the network may enable a peer-to-peer network, allowing devices coupled to the system to operate as clients or servers.

[0103] In one embodiment, the processor can execute a series of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a memory location, such as memory. The instructions can be directed to the processor, which can then be programmed or otherwise configured to implement the methods of this application. Examples of operations performed by the processor can include reading, decoding, executing, and writing back.

[0104] In one implementation, the processor may be part of a circuit such as an integrated circuit, and one or more other components of the system may be included in the circuit, which in some cases is an application-specific integrated circuit (ASIC).

[0105] In one implementation, the electronic storage unit can store files such as drivers, libraries, and saved programs. The electronic storage unit can also store user data, such as user preferences and user programs. In some cases, the system may include one or more additional data storage units located outside the computer system, such as on a remote server that communicates with the system via an intranet or the Internet.

[0106] In one implementation, the system can communicate with one or more remote computer systems via a network. For example, the system can communicate with a user's (e.g., a physician's) remote computer system. Examples of remote computer systems include personal computers, tablet PCs, telephones, smartphones, or personal digital assistants. Users can access the system via the network.

[0107] The present invention provides an electronic device, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to perform the steps of the method described above.

[0108] In one embodiment, the electronic device is a computer device, which may be a terminal, including a processor and memory connected via a system bus; it also includes a network interface, a display screen, and an input device. The processor of the computer device provides computing and control capabilities. The memory of the computer device includes non-volatile storage media and internal memory. The non-volatile storage media stores the operating system and computer programs. The internal memory provides an environment for the operation of the operating system and computer programs in the non-volatile storage media. The network interface of the computer device is used for communication with external terminals via a network connection. The display screen of the computer device may be a liquid crystal display screen or an e-ink display screen. The input device of the computer device may be a touch layer covering the display screen, or buttons, a trackball, or a touchpad mounted on the casing of the computer device, or an external keyboard, touchpad, or mouse, etc.

[0109] The above-mentioned equipment is only a part of the structure related to the present application and does not constitute a limitation on the computer equipment on which the present application is applied. The specific computer equipment may include more or fewer components than shown in the figure, or combine certain components, or have different component arrangements.

[0110] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0111] Example

[0112] The primer screening process is as follows Figure 1 As shown.

[0113] 1. Specimen Source

[0114] The study focused on 92 submitted samples from 2022 to June 2023, spanning 30 provinces. To ensure representativeness, a carefully designed sample set was used, based on the proportion of data from each province. This meticulously crafted sample set included four samples from Anhui, Fujian, Jiangsu, Qinghai, and Chongqing; three samples from Beijing, Gansu, Guangdong, Guangxi, Guizhou, Hebei, Henan, Heilongjiang, Hubei, Hunan, Jilin, Jiangxi, Liaoning, Inner Mongolia, Shandong, Shanxi, Shaanxi, Shanghai, Sichuan, Tianjin, Yunnan, and Zhejiang; and two samples each from Hainan, Ningxia, and Xinjiang. This detailed sample distribution ensured comprehensive coverage of influenza virus samples across the country, providing reliable and comprehensive foundational data for the study.

[0115] 2. Nucleic acid extraction

[0116] For the respiratory specimens received from the first confirmed case, total viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) following the instructions, and stored at -80°C for later use.

[0117] 3. Whole genome sequencing

[0118] Whole-genome-specific amplification of extracted viral total RNA was performed using FluA universal amplification primers and a one-step amplification enzyme system. PCR products were purified using the QIAquick PCR Purification kit (Qiagen, Germany). DNA sequencing libraries were constructed according to the Nextera XT DNA Library Preparation kit (Illumina, USA), and whole-genome deep sequencing was performed using a Minisq sequencer on the Illumina sequencing platform. The raw sequencing data were assembled using CLCGenomics Workbench (Version 20.0) software.

[0119] 4. PCR primer design

[0120] First, nucleic acid extraction, library construction, sequencing, and data analysis were performed on viral strain samples submitted from various provinces and cities across China, resulting in 755 identical sequences. Sequence alignment was performed using the Nextclade website, and homology analysis was conducted using Geneious Prime software. The aligned sequences were then compared with the original primer sequences. 98.55% (744 / 755) of the sequences showed mutations at site 308 (TC), and 97.62% (737 / 755) showed mutations at site 357 (TA). Based on this characteristic, a first pair of new primers was designed. Furthermore, mutations were found at sites 317 (AG), 364 (AG), and 389 (AG) in a significant number of provinces. Figure 2 Three pairs of primers were designed for these three sites.

[0121] Based on the geographical locations of the three different variant sites, after map marking, it was found that variant site 317 is mainly concentrated in southern China, with Guizhou Province being the most prominent and Guangdong Province being the second most prominent; variant site 364 is mainly concentrated in central and northern cities of China, with Sichuan Province being the most prominent and Inner Mongolia Autonomous Region being the second most prominent; and variant site 389 is mainly concentrated in coastal cities, with Fujian Province, Jiangsu Province, Shanghai Municipality and Shandong Province being the most prominent.

[0122] Using 92 samples drawn from nationwide submissions, experiments were conducted to validate the old primers and four pairs of new primers. The results showed that no significant geographical distribution was observed after altering the 317, 364, and 389 sites. The CT values ​​did not exhibit any regular changes.

[0123] The four new primer pairs with altered individual base designs did not show significant changes in sensitivity. Therefore, based on the first new primer pair, four more primer pairs were designed by arranging and combining the three mutated base sites (317, 364, and 389), including simultaneous alterations of sites 317 and 364, simultaneous alterations of sites 317 and 389, simultaneous alterations of sites 364 and 389, and simultaneous alterations of sites 317, 364, and 389. Degenerate bases were then introduced into the design based on these.

[0124] Following the single-base site design method, seven more primer pairs were successfully derived through permutation and combination. In total, this study meticulously designed 15 primer pairs, such as... Figure 3 As shown. This series of primers was designed to improve the accuracy and sensitivity of identification, laying a solid foundation for subsequent experimental research.

[0125] The specific primer design method is as follows:

[0126] (1) The subtype was chosen as H3.

[0127] (2) Primer design for H3 subtype identification: The HA segment is used as the target gene for primer design.

[0128] (3) Use tools (such as MUFFT, muscle, etc.) to align the downloaded H3 subtype fragments and generate an alignment file.

[0129] (4) Use the software Geneious Primer to check the matching degree of the existing primers in the alignment file generated in the previous step.

[0130] (5) After the program ends, an overview diagram with mismatch information will appear. The ideal primer result is mismatch=0. Therefore, based on the number of mismatches and their distribution positions in the primers, primers with single base changes, multiple base changes and degenerate bases are designed respectively.

[0131] (6) Repeat step (3) using the modified primers to verify whether it meets expectations (the number of mismatches is reduced).

[0132] (7) Experimental verification: QPCR experiments were conducted on H3 subtype influenza viruses nationwide. The results were summarized and analyzed, and it was found that by reducing mismatch sites in the same sample, the CT value could be effectively reduced based on the original primers. Among the designed primers, the most suitable primers were selected. These primers can effectively detect strains from different geographical locations and reduce their CT values.

[0133] Table 1. 16 Primer Sequences

[0134]

[0135] 5. Detection Methods

[0136] (1) Sample preparation: 92 original samples with sufficient volume (volume > 1 ml, Tianlong nucleic acid extraction uses 200 μl).

[0137] (2) Experimental reagents: Tianlong nucleic acid extraction, FastVirus one-step method, Tianlong PCR instrument.

[0138] (3) Dilute the concentration of 16 primer pairs to 10 μmol.

[0139] (4) Configuration system (Table 2).

[0140] Table 2 Configuration System

[0141]

[0142] (5) Mix the prepared system well and add it to a 96-well plate, adding 20 μl to each well.

[0143] (6) Add the sample as shown in Table 3. The total volume of each well is 25 μl, and N represents different primers.

[0144] Table 3 Different sample and primer mixtures

[0145]

[0146] (7) Use a high-permeability membrane to seal the 96-well plate.

[0147] (8) Mix well and centrifuge for 1 min.

[0148] (9) Start the instrument: Place the 96-well plate into the real-time PCR instrument and run the program.

[0149] (10) Amplification procedure:

[0150] ① Create a new program and select the fluorescence channel (FAM) based on the fluorescent label of the probe.

[0151] ② Set the reaction conditions, as shown in Table 4.

[0152] Table 4 Reaction conditions

[0153]

[0154] 6. Primer CT value pattern

[0155] Ninety-two samples from 30 provinces and municipalities across the country were tested using the qPCR method, resulting in 1472 CT value results. These 1472 experimental results were meticulously categorized, and the maximum value (represented by the largest CT value) and minimum value (represented by the smallest CT value) for each reaction system were statistically analyzed, yielding a series of data.

[0156] Through comprehensive analysis of the results of different primers ( Figure 4 The study found that primers 5 and 12 had the highest number of minimum values ​​under amplification conditions, with 22 and 24 respectively, accounting for 23.91% and 26.08% of the total. Detailed statistical analysis of the maximum and minimum values ​​revealed that, among the 16 primer pairs, primers 5 and 12 were significantly more effective in reducing CT values ​​compared to other primers. This result provides strong experimental evidence for primer selection and use.

[0157] 7. Mean between primers

[0158] Given that the H3 identification primers currently provided by the domestic influenza surveillance network are primer 0, using primer 0 as the baseline, the differences between 15 primer pairs and primer 0 were calculated, and the arithmetic mean and variance of these differences were calculated. Figure 5 As shown, from the perspective of arithmetic mean, the differences between six primer pairs (primers 3, 5, 6, 10, 12, and 15) all exceeded 1, specifically 1.17, 1.23, 1.12, 1.15, 1.37, and 1.18, respectively. Among these six primer pairs, primer 12 had the largest difference, with an arithmetic mean of 1.37, followed by primer 5 with an arithmetic mean of 1.23. Therefore, primers 12 and 5 showed the most significant reduction in CT values.

[0159] From a variance perspective, primer 5 has a variance of 2.17, while primer 12 has a variance of 0.82. This result shows that primer 12 outperforms primer 5 in terms of variance. In summary, through a combined analysis of the arithmetic mean and variance, primer 12 is found to be significantly superior to primer 5 in reducing CT values.

[0160] Discrete distribution of 8 primers

[0161] Discrete variance analysis was performed on the differences between primers 1, 5, and 12 and primer 0. Figure 6 The data clearly shows the wide distribution of data points across the entire coordinate axis. Primer 1 exhibits the highest numerical aggregation, with values ​​all near zero, indicating that the difference in primer 1 does not vary significantly and remains at a relatively stable level. Conversely, primer 5 shows greater dispersion, with both positive and negative biases. The trend of data fluctuating around zero indicates that primer 5 exhibits instability in some situations. Most values ​​for primer 12 are above zero, showing an overall positive trend. This demonstrates that primer 12 has a significant effect on reducing CT values, showcasing its good performance.

[0162] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A primer set for detecting H3 influenza virus, characterized in that, The primer set includes the primer pairs shown in SEQ ID NO:10 and 12, the primer pairs shown in SEQ ID NO:16 and 18, the primer pairs shown in SEQ ID NO:19 and 21, the primer pairs shown in SEQ ID NO:31 and 33, the primer pairs shown in SEQ ID NO:37 and 39, and the primer pairs shown in SEQ ID NO:46 and 48.

2. The primer set according to claim 1, characterized in that, The primer set also includes probes, the nucleotide sequences of which are shown in SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:38 and SEQ ID NO:

47.

3. The primer set according to claim 1, characterized in that, The primer set includes a forward primer and a reverse primer; at least one of the forward primer and the reverse primer is labeled with a labeling substance.

4. The primer set according to claim 3, characterized in that, The labeling substance is a fluorescent substance, a radioactive isotope, or an enzyme.

5. The primer set according to claim 4, characterized in that, The fluorescent substance is TAMRAT. TM Alexa555, Alexa647, Cy3, Cy5, or fluorescein.

6. The primer set according to claim 4, characterized in that, The radioactive isotope is 32 P, 33 P or 35 S.

7. The primer set according to claim 4, characterized in that, The enzyme is alkaline phosphatase or horseradish peroxidase.

8. A kit for detecting H3 influenza virus in a sample, characterized in that, The kit comprises the primer set as described in any one of claims 1-7.

9. The reagent kit according to claim 8, characterized in that, The kit also includes enzymes, dNTPs, buffer solutions, or Mg2+ for PCR. 2+ At least one of the reaction reagents.

10. The reagent kit according to claim 9, characterized in that, The enzymes used for PCR are DNA polymerase and / or RNA polymerase.

11. The reagent kit according to claim 9, characterized in that, The buffer solution is either polymerase buffer or Tris hydrochloric acid buffer.

12. The reagent kit according to claim 8, characterized in that, The kit also includes instructions.

13. A method for detecting H3 influenza virus in a sample for non-diagnostic purposes, characterized in that, The method includes amplification using the primer set according to any one of claims 1-7.

14. The use of the primer set according to any one of claims 1-7 in the preparation of a kit for detecting H3 influenza virus.

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